Although they are usually studied with purified components in dilute solution, protein-nucleic acid interactions are very sensitive to changes in physiologically significant environmental variables. We have initiated a study of the effects of varying the solution osmolarity (oncotic pressure) on the interaction of gene regulatory proteins with DNA, using the gal repressor protein interaction with its operator sequence as a model system. The osmotic pressure of the reaction is varied by adding high concentrations of different solutes (osmoticant). Solutes which are excluded from the surfaces of both the DNA and protein (e.g..polyethylene glycol, glycine betaine) drastically enhance complex formation. Solutes which are not excluded from one of the surfaces appear to have little or no effect, indicating that this is not simply a water activity phenomenon. We have interpreted these results in terms of the volume of the solution inaccessible to the solute molecule which changes upon the protein binding to the DNA. This allows one to determine the total number of water molecules released from the surfaces of both the protein and DNA upon complexation, of the area involved in the reaction, which should be related to the amount of surface area of both protein and DNA occluded in the complex. Such insight into the hydration of both surfaces before and after complexation is important in order to understand the stability of a sequence-specific protein-DNA complex.